PROJECT SUMMARY: Kidney stone (KS) disease is becoming more prevalent in the United States and is associated with systemic diseases and lifestyle factors such as diet. A major risk factor for stone formation is an elevation in urinary oxalate, which can be derived endogenously or from the diet. Dietary oxalate intake may induce supersaturation of calcium oxalate (CaOx) which may generate crystals of this stone forming salt in urine and perhaps the nephron. CaOx crystals that interact with renal epithelium activate innate immunity by releasing cytokines and chemokines to stimulate monocyte recruitment. We previously reported that patients with CaOx kidney stone (CaOx KS) disease have altered monocyte cellular energetics and increased inflammation. In addition, we have shown oxalate directly disrupts cellular energetics and redox homeostasis in monocytes from healthy subjects. The long-term goal of this research is to understand how oxalate impacts monocyte and macrophage immunometabolism in KS disease. The objective of the current study is to investigate the relationship between urinary crystals, macrophage activation, reactive oxygen species (ROS), and pro- inflammatory signaling pathways following oxalate consumption. The central hypothesis is oxalate disrupts mitochondrial complex I activity in monocytes and stimulates ROS generation, pro-inflammatory macrophage differentiation, and renal crystal deposition in CaOx KS disease. Aim 1 will test the hypothesis that oxalate stimulates reverse electron transport (RET) through mitochondrial complex I in monocytes from healthy subjects. Aim 2 will test the hypothesis that oxalate mediated crystalluria induces ROS and disrupts macrophage immunometabolism in rat kidneys. No studies to date have investigated the connection between dietary oxalate, crystalluria, and immune responses in CaOx KS disease. A novel approach to assess urinary nanocrystals and their influence on monocytes and macrophages will be investigated. The impact of this research will help us understand how monocytes and macrophages respond to crystals, and may identify potential approaches to assess stone risk, reduce stone formation, and stone recurrence. The data generated from this proposal will advance our knowledge about the role of dietary oxalate on immune cells in KS disease and serve as a key foundation for subsequent R01 funding.